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Direct evidence of conformational changes associated with voltage gating in a voltage sensor protein by time-resolved X-ray/neutron interferometry.

Tronin AY, Nordgren CE, Strzalka JW, Kuzmenko I, Worcester DL, Lauter V, Freites JA, Tobias DJ, Blasie JK - Langmuir (2014)

Bottom Line: Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes.Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance.The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD's atomic-level 3-D structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.

ABSTRACT
The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD's profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD's atomic-level 3-D structure.

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Left side: comparison of the difference xSLD profilesdetermined from the X-ray interferometry experiments for VSD:POPCmembrane tethered to the surface of an inorganic multilayer substrate(dotted) from the first cycle of the series of potentials applied,with those calculated from molecular dynamics simulations for an untetheredVSD:POPC membrane (solid) for the two pairs of potentials indicated,each membrane fully hydrated. Right side: the same comparison, butwith the abscissa scale for the difference profilescalculated from the simulation expanded by 25%. The time-averagedprofiles calculated from the simulations have been smoothed via convolutionwith a Gaussian whose 1/e width was 2σ.
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fig6: Left side: comparison of the difference xSLD profilesdetermined from the X-ray interferometry experiments for VSD:POPCmembrane tethered to the surface of an inorganic multilayer substrate(dotted) from the first cycle of the series of potentials applied,with those calculated from molecular dynamics simulations for an untetheredVSD:POPC membrane (solid) for the two pairs of potentials indicated,each membrane fully hydrated. Right side: the same comparison, butwith the abscissa scale for the difference profilescalculated from the simulation expanded by 25%. The time-averagedprofiles calculated from the simulations have been smoothed via convolutionwith a Gaussian whose 1/e width was 2σ.

Mentions: Prior to addressing the structural significanceof the observedpotential-dependent changes in the SLD profiles, it is important toreiterate that the changes described were both reproducible and reversible.Reproducibility for both the X-ray and neutron experiments is describedin some detail in the Supporting Information. The “inactivation” associated with the complete KvAPchannel (from which the VSD was derived) in electrophysiological experiments,27 arising from multiple cycles of depolarizationand repolarization of the membrane potential on a time scale of 100–200ms per potential, could have undermined these experiments. In theX-ray case, it was not necessary to average over multiple cycles,and the experimental modulus data for the two 0 mV potentials employedwithin each of the four cycles investigated superimposed to withinthe noise level, demonstrating reversibility despite the longer timescale of 5–20 s per potential. Furthermore, the potential-dependentchanges in the xSLD profiles were similar for the first and secondcycles, in terms of the positions and amplitudes of their major featuresas evident from a comparison of Figures 3 and 6, although they do differ in detail. Neither wouldhave occurred with any substantial level of inactivation comparableto that for the complete KvAP channel, for which the entire ensemblebecomes inactivated after only three cycles.27 For the neutron case, averaging over many cycles was required toachieve a satisfactory noise level in the modulus data for each valueof the potential. Again, if inactivation on a time scale of 20 s,comparable to that for the complete KvAP channel, had been a majorfactor cycle-to-cycle, potential-dependent changes in the modulusdata would have vanished with averaging over many cycles. Thus, theinactivation associated with the complete channel does not appearto play any significant role in these studies of the isolated VSDprotein.


Direct evidence of conformational changes associated with voltage gating in a voltage sensor protein by time-resolved X-ray/neutron interferometry.

Tronin AY, Nordgren CE, Strzalka JW, Kuzmenko I, Worcester DL, Lauter V, Freites JA, Tobias DJ, Blasie JK - Langmuir (2014)

Left side: comparison of the difference xSLD profilesdetermined from the X-ray interferometry experiments for VSD:POPCmembrane tethered to the surface of an inorganic multilayer substrate(dotted) from the first cycle of the series of potentials applied,with those calculated from molecular dynamics simulations for an untetheredVSD:POPC membrane (solid) for the two pairs of potentials indicated,each membrane fully hydrated. Right side: the same comparison, butwith the abscissa scale for the difference profilescalculated from the simulation expanded by 25%. The time-averagedprofiles calculated from the simulations have been smoothed via convolutionwith a Gaussian whose 1/e width was 2σ.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4007984&req=5

fig6: Left side: comparison of the difference xSLD profilesdetermined from the X-ray interferometry experiments for VSD:POPCmembrane tethered to the surface of an inorganic multilayer substrate(dotted) from the first cycle of the series of potentials applied,with those calculated from molecular dynamics simulations for an untetheredVSD:POPC membrane (solid) for the two pairs of potentials indicated,each membrane fully hydrated. Right side: the same comparison, butwith the abscissa scale for the difference profilescalculated from the simulation expanded by 25%. The time-averagedprofiles calculated from the simulations have been smoothed via convolutionwith a Gaussian whose 1/e width was 2σ.
Mentions: Prior to addressing the structural significanceof the observedpotential-dependent changes in the SLD profiles, it is important toreiterate that the changes described were both reproducible and reversible.Reproducibility for both the X-ray and neutron experiments is describedin some detail in the Supporting Information. The “inactivation” associated with the complete KvAPchannel (from which the VSD was derived) in electrophysiological experiments,27 arising from multiple cycles of depolarizationand repolarization of the membrane potential on a time scale of 100–200ms per potential, could have undermined these experiments. In theX-ray case, it was not necessary to average over multiple cycles,and the experimental modulus data for the two 0 mV potentials employedwithin each of the four cycles investigated superimposed to withinthe noise level, demonstrating reversibility despite the longer timescale of 5–20 s per potential. Furthermore, the potential-dependentchanges in the xSLD profiles were similar for the first and secondcycles, in terms of the positions and amplitudes of their major featuresas evident from a comparison of Figures 3 and 6, although they do differ in detail. Neither wouldhave occurred with any substantial level of inactivation comparableto that for the complete KvAP channel, for which the entire ensemblebecomes inactivated after only three cycles.27 For the neutron case, averaging over many cycles was required toachieve a satisfactory noise level in the modulus data for each valueof the potential. Again, if inactivation on a time scale of 20 s,comparable to that for the complete KvAP channel, had been a majorfactor cycle-to-cycle, potential-dependent changes in the modulusdata would have vanished with averaging over many cycles. Thus, theinactivation associated with the complete channel does not appearto play any significant role in these studies of the isolated VSDprotein.

Bottom Line: Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes.Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance.The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD's atomic-level 3-D structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Pennsylvania , Philadelphia, Pennsylvania 19104, United States.

ABSTRACT
The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD's profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD's atomic-level 3-D structure.

Show MeSH
Related in: MedlinePlus